Methods of administering anti-tnfalpha  antibodies

ABSTRACT

Methods of treating disorders in which TNFα activity is detrimental via biweekly, subcutaneous administration of human antibodies, preferably recombinant human antibodies, that specifically bind to human tumor necrosis factor α (hTNFα) are disclosed. The antibody may be administered with or without methotrexate. These antibodies have high affinity for hTNFα (e.g., K d =10 −8  M or less), a slow off rate for hTNFα dissociation (e.g., K off =10 −3  sec −1  or less) and neutralize hTNFα activity in vitro and in vivo. An antibody of the invention can be a full-length antibody or an antigen-binding portion thereof. Kits containing a pharmaceutical composition and instructions for dosing, and preloaded syringes containing pharmaceutical compositions are also encompassed by the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.10/163,657, filed on Jun. 5, 2002, which claims the benefit of U.S.Provisional Application No. 60/296,961, filed Jun. 8, 2001. Each ofthese applications is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Tumor necrosis factor α (TNFα) is a cytokine produced by numerous celltypes, including monocytes and macrophages, that was originallyidentified based on its capacity to induce the necrosis of certain mousetumors (see e.g., Old, L. (1985) Science 230:630-632). Subsequently, afactor termed cachectin, associated with cachexia, was shown to be thesame molecule as TNFα. TNFα has been implicated in mediating shock (seee.g., Beutler, B. and Cerami, A. (1988) Annu. Rev. Biochem. 57:505-518;Beutler, B. and Cerami, A. (1989) Annu. Rev. Immunol. 7:625-655).Furthermore, TNFα has been implicated in the pathophysiology of avariety of other human diseases and disorders, including sepsis,infections, autoimmune diseases, transplant rejection andgraft-versus-host disease (see e.g., Vasilli, P. (1992) Annu. Rev.Immunol. 10:411-452; Tracey, K. J. and Cerami, A. (1994) Annu. Rev. Med.45:491-503).

Because of the harmful role of human TNFα (hTNFα) in a variety of humandisorders, therapeutic strategies have been designed to inhibit orcounteract hTNFα activity. In particular, antibodies that bind to, andneutralize, hTNFα have been sought as a means to inhibit hTNFα activity.Some of the earliest of such antibodies were mouse monoclonal antibodies(mAbs), secreted by hybridomas prepared from lymphocytes of miceimmunized with hTNFα (see e.g., Hahn T; et al., (1985) Proc Natl AcadSci USA 82: 3814-3818; Liang, C-M., et al. (1986) Biochem. Biophys. Res.Commun. 137:847-854; Hirai, M., et al. (1987) J. Immunol. Methods96:57-62; Fendly, B. M., et al. (1987) Hybridoma 6:359-370; Möller, A.,et al. (1990) Cytokine 2:162-169; U.S. Pat. No. 5,231,024 to Moeller etal.; European Patent Publication No. 186 833 B1 by Wallach, D.; EuropeanPatent Application Publication No. 218 868 A1 by Old et al.; EuropeanPatent Publication No. 260 610 B1 by Moeller, A., et al.). While thesemouse anti-hTNFα antibodies often displayed high affinity for hTNFα(e.g., K_(d)≦10⁻⁹M) and were able to neutralize hTNFα activity, theiruse in vivo may be limited by problems associated with administration ofmouse antibodies to humans, such as short serum half life, an inabilityto trigger certain human effector functions and elicitation of anunwanted immune response against the mouse antibody in a human (the“human anti-mouse antibody” (HAMA) reaction).

In an attempt to overcome the problems associated with use offully-murine antibodies in humans, murine anti-hTNFα antibodies havebeen genetically engineered to be more “human-like.” For example,chimeric antibodies, in which the variable regions of the antibodychains are murine-derived and the constant regions of the antibodychains are human-derived, have been prepared (Knight, D. M, et al.(1993) Mol. Immunol. 30:1443-1453; PCT Publication No. WO 92/16553 byDaddona, P. E., et al.). Additionally, humanized antibodies, in whichthe hypervariable domains of the antibody variable regions aremurine-derived but the remainder of the variable regions and theantibody constant regions are human-derived, have also been prepared(PCT Publication No. WO 92/11383 by Adair, J. R., et al.). However,because these chimeric and humanized antibodies still retain some murinesequences, they still may elicit an unwanted immune reaction, the humananti-chimeric antibody (HACA) reaction, especially when administered forprolonged periods, e.g., for chronic indications, such as rheumatoidarthritis (see e.g., Elliott, M. J., et al. (1994) Lancet 344:1125-1127;Elliot, M. J., et al. (1994) Lancet 344:1105-1110).

A preferred hTNFα inhibitory agent to murine mAbs or derivatives thereof(e.g., chimeric or humanized antibodies) would be an entirely humananti-hTNFα antibody, since such an agent should not elicit the HAMAreaction, even if used for prolonged periods. Human monoclonalautoantibodies against hTNFα have been prepared using human hybridomatechniques (Boyle, P., et al. (1993) Cell. Immunol. 152:556-568; Boyle,P., et al. (1993) Cell. Immunol. 152:569-581; European PatentApplication Publication No. 614 984 A2 by Boyle, et al.). However, thesehybridoma-derived monoclonal autoantibodies were reported to have anaffinity for hTNFα that was too low to calculate by conventionalmethods, were unable to bind soluble hTNFα and were unable to neutralizehTNFα-induced cytotoxicity (see Boyle, et al.; supra). Moreover, thesuccess of the human hybridoma technique depends upon the naturalpresence in human peripheral blood of lymphocytes producingautoantibodies specific for hTNFα. Certain studies have detected serumautoantibodies against hTNFα in human subjects (Fomsgaard, A., et al.(1989) Scand. J. Immunol. 30:219-223; Bendtzen, K., et al. (1990) Prog.Leukocyte Biol. 10B:447-452), whereas others have not (Leusch, H- G., etal. (1991) J. Immunol. Methods 139:145-147).

Alternative to naturally-occurring human anti-hTNFα antibodies would bea recombinant hTNFα antibody. Recombinant human antibodies that bindhTNFα with relatively low affinity (i.e., K_(d)˜10⁻⁷M) and a fast offrate (i.e., K_(off)˜10⁻² sec⁻¹) have been described (Griffiths, A. D.,et al. (1993) EMBO J. 12:725-734). However, because of their relativelyfast dissociation kinetics, these antibodies may not be suitable fortherapeutic use. Additionally, a recombinant human anti-hTNFα has beendescribed that does not neutralize hTNFα activity, but rather enhancesbinding of hTNFα to the surface of cells and enhances internalization ofhTNFα (Lidbury, A., et al. (1994) Biotechnol. Ther. 5:27-45; PCTPublication No. WO 92/03145 by Aston, R. et al.)

Recombinant human antibodies that bind soluble hTNFα with high affinityand slow dissociation kinetics and that have the capacity to neutralizehTNFα activity, including hTNFα-induced cytotoxicity (in vitro and invivo) and hTNFα-induced cell activation, have also been described (seeU.S. Pat. No. 6,090,382). Typical protocols for administering antibodiesare performed intravenously on a weekly basis. Weekly dosing withantibodies and/or any drug can be costly, cumbersome, and result in anincrease in the number of side effects due to the frequency ofadministration. Intravenous administration also has limitations in thatthe administration is usually provided by someone with medical training.

SUMMARY OF THE INVENTION

The present invention provides methods for biweekly dosing regimens forthe treatment of TNFα associated disorders, preferably via asubcutaneous route. Biweekly dosing has many advantages over weeklydosing including, but not limited to, a lower number of totalinjections, decreased number of injection site reactions (e.g., localpain and swelling), increased patient compliance (i.e., due to lessfrequent injections), and less cost to the patient as well as the healthcare provider. Subcutaneous dosing is advantageous because the patientmay self-administer a therapeutic substance, e.g., a human TNFαantibody, which is convenient for both the patient and the health careprovider.

This invention provides methods for treating disorders in which TNFαactivity is detrimental. The methods include administering biweekly,subcutaneous injections of antibodies to a subject. The antibodiespreferably are recombinant human antibodies that specifically bind tohuman TNFα. This invention further provides methods for treatingdisorders in which TNFα activity is detrimental. These methods includeutilizing a combination therapy wherein human antibodies areadministered to a subject with another therapeutic agent, such as one ormore additional antibodies that bind other targets (e.g., antibodiesthat bind other cytokines or that bind cell surface molecules), one ormore cytokines, soluble TNFα receptor (see e.g., PCT Publication No. WO94/06476) and/or one or more chemical agents that inhibit hTNFαproduction or activity (such as cyclohexane-ylidene derivatives asdescribed in PCT Publication No. WO 93/19751), preferably methotrexate.The antibodies are preferably recombinant human antibodies thatspecifically bind to human TNFα. The antibodies of the invention arecharacterized by binding to hTNFα with high affinity and slowdissociation kinetics and by neutralizing hTNFα activity, includinghTNFα-induced cytotoxicity (in vitro and in vivo) and hTNFα-inducedcellular activation. The antibodies can be full-length (e.g., an IgG1 orIgG4 antibody) or can comprise only an antigen-binding portion (e.g., aFab, F(ab′)₂, scFv fragment or single domain). The most preferredrecombinant antibody of the invention, termed D2E7, has a light chainCDR3 domain comprising the amino acid sequence of SEQ ID NO: 3 and aheavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:4 (set forth in Appendix B). Preferably, the D2E7 antibody has a lightchain variable region (LCVR) comprising the amino acid sequence of SEQID NO: 1 and a heavy chain variable region (HCVR) comprising the aminoacid sequence of SEQ ID NO: 2. These antibodies are described in U.S.Pat. No. 6,090,382, incorporated in its entirety herein by reference.

In one embodiment, the invention provides methods of treating disordersin which TNFα activity is detrimental. These methods include inhibitinghuman TNFα activity by subcutaneous, biweekly administration of ananti-TNFα antibody such that the disorder is treated. The disorder canbe, for example, sepsis, an autoimmune disease (e.g., rheumatoidarthritis, allergy, multiple sclerosis, autoimmune diabetes, autoimmuneuveitis and nephrotic syndrome), an infectious disease, a malignancy,transplant rejection or graft-versus-host disease, a pulmonary disorder,a bone disorder, an intestinal disorder or a cardiac disorder.

In another embodiment, the invention provides methods of treatingdisorders in which TNFα activity is detrimental. These methods includeinhibiting human TNFα activity by subcutaneous administration of ananti-TNFα antibody and methotrexate such that the disorder is treated.In one aspect, methotrexate is administered together with an anti-TNFαantibody. In another aspect, methotrexate is administered prior to theadministration of an anti-TNFα antibody. In still another aspect,methotrexate is administered subsequent to the administration of ananti-TNFα antibody.

In a preferred embodiment, the anti-TNFα antibody used to treatdisorders in which TNFα activity is detrimental is a human anti-TNFαantibody. Even more preferably, treatment occurs by the biweekly,subcutaneous administration of an isolated human antibody, or anantigen-binding portion thereof. The antibody or antigen-binding portionthereof preferably dissociates from human TNFα with a K_(d) of 1×10⁻⁸ Mor less and a K_(off) rate constant of 1×10⁻³ s⁻¹ or less, bothdetermined by surface plasmon resonance, and neutralizes human TNFαcytotoxicity in a standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁷ Mor less. More preferably, the isolated human antibody, orantigen-binding portion thereof, dissociates from human TNFα with aK_(off) of 5×10⁻⁴ s⁻¹ or less, or even more preferably, with a K_(off)of 1×10⁻⁴ s⁻¹ or less. More preferably, the isolated human antibody, orantigen-binding portion thereof, neutralizes human TNFα cytotoxicity ina standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁸ M or less, evenmore preferably with an IC₅₀ of 1×10⁻⁹ M or less and still morepreferably with an IC₅₀ of 1×10⁻¹⁰ M or less.

In another embodiment, the invention provides methods of treatingdisorders in which TNFα activity is detrimental by the biweekly,subcutaneous administration to the subject a human antibody, orantigen-binding portion thereof. The antibody or antigen-binding portionthereof preferably has the following characteristics:

a) dissociates from human TNFα with a K_(off) of 1×10⁻³ s⁻¹ or less, asdetermined by surface plasmon resonance;

b) has a light chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alaninesubstitution at position 1, 4, 5, 7 or 8 or by one to five conservativeamino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9;

c) has a heavy chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alaninesubstitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to fiveconservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9,10, 11 and/or 12.

More preferably, the antibody, or antigen-binding portion thereof,dissociates from human TNFα with a K_(off) of 5×10⁻⁴ s⁻¹ or less. Stillmore preferably, the antibody, or antigen-binding portion thereof,dissociates from human TNFα with a K_(off) of 1×10⁻⁴ s⁻¹ or less.

In yet another embodiment, the invention provides methods of treatingdisorders in which TNFα activity is detrimental. These methods include abiweekly, subcutaneous administration to the subject a human antibody,or an antigen-binding portion thereof. The antibody or antigen-bindingportion thereof preferably contains an LCVR having CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 3, or modified from SEQID NO: 3 by a single alanine substitution at position 1, 4, 5, 7 or 8,and with an HCVR having a CDR3 domain comprising the amino acid sequenceof SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alaninesubstitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11. More preferably,the LCVR further has a CDR2 domain comprising the amino acid sequence ofSEQ ID NO: 5 and the HCVR further has a CDR2 domain comprising the aminoacid sequence of SEQ ID NO: 6. Still more preferably, the LCVR furtherhas CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7 andthe HCVR has a CDR1 domain comprising the amino acid sequence of SEQ IDNO: 8.

In still another embodiment, the invention provides methods of treatingdisorders in which TNFα activity is detrimental by subcutaneouslyadministering to the subject, biweekly, an isolated human antibody, oran antigen binding portion thereof. The antibody or antigen-bindingportion thereof preferably contains an LCVR comprising the amino acidsequence of SEQ ID NO: 1 and an HCVR comprising the amino acid sequenceof SEQ ID NO: 2. In certain embodiments, the antibody has an IgG1 heavychain constant region or an IgG4 heavy chain constant region. In yetother embodiments, the antibody is a Fab fragment, an F(ab′)₂ fragmentor a single chain Fv fragment.

In still other embodiments, the invention provides methods of treatingdisorders in which the administration of an anti-TNFα antibody isbeneficial by subcutaneously administering to the subject, biweekly, oneor more anti-TNFα antibodies, or antigen-binding portions thereof. Theantibody or antigen-binding portion thereof preferably contains an LCVRhaving CDR3 domain comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ IDNO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22,SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or with anHCVR having a CDR3 domain comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28,SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO:33, SEQ ID NO: 34 and SEQ ID NO: 35.

Still another aspect of the invention pertains to kits containing aformulation comprising a pharmaceutical composition. The kits comprisean anti-TNFα antibody and a pharmaceutically acceptable carrier. Thekits contain instructions for biweekly subcutaneous dosing of thepharmaceutical composition for the treatment of a disorder in which theadministration of an anti-TNFα antibody is beneficial. In anotheraspect, the invention pertains to kits containing a formulationcomprising a pharmaceutical composition, further comprising an anti-TNFαantibody, methotrexate, and a pharmaceutically acceptable carrier. Thekits contain instructions for subcutaneous dosing of the pharmaceuticalcomposition for the treatment of a disorder in which the administrationof an anti-TNFα antibody is beneficial.

Still another aspect of the invention provides a preloaded syringecontaining a pharmaceutical composition comprising an anti-TNFα antibodyand a pharmaceutically acceptable carrier. In still another aspect, theinvention provides a preloaded syringe containing a pharmaceuticalcomposition comprising an anti-TNFα antibody, methotrexate, and apharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the American College of Rheumatology 20 (ACR20) and ACR50responses for patients suffering from rheumatoid arthritis (RA) aftersubcutaneous dosing with the antibody D2E7 every week for a total oftwelve weeks (top), or subcutaneous dosing with the antibody D2E7 andmethotrexate every other week (bottom) for a total of twenty-four weeks.These data indicate that every other week dosing is as effective asevery week dosing.

FIG. 2 depicts ACR20, ACR50, and ACR70 responses for patients sufferingfrom RA after subcutaneous dosing with the antibody D2E7 andmethotrexate every other week at twenty-four weeks.

FIGS. 3A and 3B depict time courses of tender joint count (3A) andswollen joint count (3B) over twenty-four weeks for patients sufferingfrom RA after subcutaneous dosing with D2E7 and methotrexate every otherweek at twenty-four weeks.

FIGS. 4A and 4B depict results from a short form health survey (SF-36)from patients suffering from RA after subcutaneous dosing with theantibody D2E7 and methotrexate every other week at twenty-four weeks.RP, role physical; PF, physical function; BP, bodily pain; GH, generalhealth; V, vitality; SF, social functioning; RE, role emotional; and ME,mental health.

FIG. 5 depicts the percentage of ACR responders following a singleintravenous injection of the antibody D2E7 and methotrexate in patientssuffering from RA.

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to methods of treating disorders in which theadministration of an anti-TNFα antibody is beneficial comprising theadministration of isolated human antibodies, or antigen-binding portionsthereof, that bind to human TNFα with high affinity, a low off rate andhigh neutralizing capacity such that the disorder is treated. Variousaspects of the invention relate to treatment with antibodies andantibody fragments, and pharmaceutical compositions thereof.

In order that the present invention may be more readily understood,certain terms are first defined.

The term “dosing”, as used herein, refers to the administration of asubstance (e.g., an anti-TNFα antibody) to achieve a therapeuticobjective (e.g., the treatment of a TNFα-associated disorder).

The terms “biweekly dosing regimen”, “biweekly dosing”, and “biweeklyadministration”, as used herein, refer to the time course ofadministering a substance (e.g., an anti-TNFα antibody) to a subject toachieve a therapeutic objective (e.g., the treatment of aTNFα-associated disorder). The biweekly dosing regimen is not intendedto include a weekly dosing regimen. Preferably, the substance isadministered every 9-19 days, more preferably, every 11-17 days, evenmore preferably, every 13-15 days, and most preferably, every 14 days.

The term “combination therapy”, as used herein, refers to theadministration of two or more therapeutic substances, e.g., an anti-TNFαantibody and the drug methotrexate. The methotrexate may be administeredconcomitant with, prior to, or following the administration of ananti-TNFα antibody.

The term “human TNFα” (abbreviated herein as hTNFα, or simply hTNF), asused herein, is intended to refer to a human cytokine that exists as a17 kD secreted form and a 26 kD membrane associated form, thebiologically active form of which is composed of a trimer ofnoncovalently bound 17 kD molecules. The structure of TNFα is describedfurther in, for example, Pennica, D., et al. (1984) Nature 312:724-729;Davis, J. M., et al. (1987) Biochemistry 26:1322-1326; and Jones, E. Y.,et al. (1989) Nature 338:225-228. The term human TNFα is intended toinclude recombinant human TNFα (rhTNFα), which can be prepared bystandard recombinant expression methods or purchased commercially (R & DSystems, Catalog No. 210-TA, Minneapolis, Minn.).

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds.Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as HCVR or VH) and a heavy chain constant region.The heavy chain constant region is comprised of three domains, CH1, CH2and CH3. Each light chain is comprised of a light chain variable region(abbreviated herein as LCVR or VL) and a light chain constant region.The light chain constant region is comprised of one domain, CL. The VHand VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., hTNFαL). It has been shown that the antigen-binding function ofan antibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).

Still further, an antibody or antigen-binding portion thereof may bepart of a larger immunoadhesion molecules, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques, asdescribed herein.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther in Section II, below), antibodies isolated from a recombinant,combinatorial human antibody library (described further in Section III,below), antibodies isolated from an animal (e.g., a mouse) that istransgenic for human immunoglobulin genes (see e.g., Taylor, L. D., etal. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared,expressed, created or isolated by any other means that involves splicingof human immunoglobulin gene sequences to other DNA sequences. Suchrecombinant human antibodies have variable and constant regions derivedfrom human germline immunoglobulin sequences. In certain embodiments,however, such recombinant human antibodies are subjected to in vitromutagenesis (or, when an animal transgenic for human Ig sequences isused, in vivo somatic mutagenesis) and thus the amino acid sequences ofthe VH and VL regions of the recombinant antibodies are sequences that,while derived from and related to human germline VH and VL sequences,may not naturally exist within the human antibody germline repertoire invivo.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds hTNFα is substantially free of antibodies that specifically bindantigens other than hTNFα). An isolated antibody that specifically bindshTNFα may, however, have cross-reactivity to other antigens, such ashTNFα molecules from other species (discussed in further detail below).Moreover, an isolated antibody may be substantially free of othercellular material and/or chemicals.

A “neutralizing antibody”, as used herein (or an “antibody thatneutralized hTNFα activity”), is intended to refer to an antibody whosebinding to hTNFα results in inhibition of the biological activity ofhTNFα. This inhibition of the biological activity of hTNFα can beassessed by measuring one or more indicators of hTNFα biologicalactivity, such as hTNFα-induced cytotoxicity (either in vitro or invivo), hTNFα-induced cellular activation and hTNFα binding to hTNFαreceptors. These indicators of hTNFα biological activity can be assessedby one or more of several standard in vitro or in vivo assays known inthe art (see Example 4). Preferably, the ability of an antibody toneutralize hTNFα activity is assessed by inhibition of hTNFα-inducedcytotoxicity of L929 cells. As an additional or alternative parameter ofhTNFα activity, the ability of an antibody to inhibit hTNFα-inducedexpression of ELAM-1 on HUVEC, as a measure of hTNFα-induced cellularactivation, can be assessed.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Example 1 and Jönsson, U., et al. (1993) Ann.Biol. Clin. 51:19-26; Jönsson, U., et al. (1991) Biotechniques11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; andJohnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

The term “K_(off)”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex.

The term “K_(d)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction.

The term “nucleic acid molecule”, as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The term “isolated nucleic acid molecule”, as used herein in referenceto nucleic acids encoding antibodies or antibody portions (e.g., VH, VL,CDR3) that bind hTNFα, is intended to refer to a nucleic acid moleculein which the nucleotide sequences encoding the antibody or antibodyportion are free of other nucleotide sequences encoding antibodies orantibody portions that bind antigens other than hTNFα, which othersequences may naturally flank the nucleic acid in human genomic DNA.Thus, for example, an isolated nucleic acid of the invention encoding aVH region of an anti-hTNFα antibody contains no other sequences encodingother VH regions that bind antigens other than hTNFα.

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Human Antibodies that Bind Human TNFα

This invention provides methods of treating disorders in which theadministration of an anti-TNFα antibody is beneficial. These methodsinclude the biweekly, subcutaneous administration of isolated humanantibodies, or antigen-binding portions thereof, that bind to human TNFαwith high affinity, a low off rate and high neutralizing capacity.Preferably, the human antibodies of the invention are recombinant,neutralizing human anti-hTNFα antibodies. The most preferredrecombinant, neutralizing antibody of the invention is referred toherein as D2E7 (the amino acid sequence of the D2E7 VL region is shownin SEQ ID NO: 1; the amino acid sequence of the D2E7 VH region is shownin SEQ ID NO: 2). The properties of D2E7 have been described in Salfeldet al., U.S. Pat. No. 6,090,382, which is incorporated by referenceherein.

In one aspect, the invention pertains to treating disorders in which theadministration of an anti-TNFα antibody is beneficial. These treatmentsinclude the biweekly, subcutaneous administration of D2E7 antibodies andantibody portions, D2E7-related antibodies and antibody portions, andother human antibodies and antibody portions with equivalent propertiesto D2E7, such as high affinity binding to hTNFα with low dissociationkinetics and high neutralizing capacity. In one embodiment, theinvention provides treatment with an isolated human antibody, or anantigen-binding portion thereof, that dissociates from human TNFα with aK_(d) of 1×10⁻⁸ M or less and a K_(off) rate constant of 1×10⁻³ s⁻¹ orless, both determined by surface plasmon resonance, and neutralizeshuman TNFα cytotoxicity in a standard in vitro L929 assay with an IC₅₀of 1×10⁻⁷ M or less. More preferably, the isolated human antibody, orantigen-binding portion thereof, dissociates from human TNFα with aK_(off) of 5×10⁻⁴ s⁻¹ or less, or even more preferably, with a K_(off)of 1×10⁻⁴ s⁻¹ or less. More preferably, the isolated human antibody, orantigen-binding portion thereof, neutralizes human TNFα cytotoxicity ina standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁸ M or less, evenmore preferably with an IC₅₀ of 1×10⁻⁹ M or less and still morepreferably with an IC₅₀ of 1×10⁻¹⁰ M or less. In a preferred embodiment,the antibody is an isolated human recombinant antibody, or anantigen-binding portion thereof.

It is well known in the art that antibody heavy and light chain CDR3domains play an important role in the binding specificity/affinity of anantibody for an antigen. Accordingly, in another aspect, the inventionpertains to methods of treating disorders in which the administration ofan anti-TNFα antibody is beneficial by subcutaneous administration ofhuman antibodies that have slow dissociation kinetics for associationwith hTNFα and that have light and heavy chain CDR3 domains thatstructurally are identical to or related to those of D2E7. Position 9 ofthe D2E7 VL CDR3 can be occupied by Ala or Thr without substantiallyaffecting the K_(off). Accordingly, a consensus motif for the D2E7 VLCDR3 comprises the amino acid sequence: Q-R-Y-N-R-A-P-Y-(T/A) (SEQ IDNO: 3). Additionally, position 12 of the D2E7 VH CDR3 can be occupied byTyr or Asn, without substantially affecting the K_(off). Accordingly, aconsensus motif for the D2E7 VH CDR3 comprises the amino acid sequence:V-S-Y-L-S-T-A-S-S-L-D-(Y/N) (SEQ ID NO: 4). Moreover, as demonstrated inExample 2 of U.S. Pat. No. 6,090,382, the CDR3 domain of the D2E7 heavyand light chains is amenable to substitution with a single alanineresidue (at position 1, 4, 5, 7 or 8 within the VL CDR3 or at position2, 3, 4, 5, 6, 8, 9, 10 or 11 within the VH CDR3) without substantiallyaffecting the K_(off). Still further, the skilled artisan willappreciate that, given the amenability of the D2E7 VL and VH CDR3domains to substitutions by alanine, substitution of other amino acidswithin the CDR3 domains may be possible while still retaining the lowoff rate constant of the antibody, in particular substitutions withconservative amino acids. A “conservative amino acid substitution”, asused herein, is one in which one amino acid residue is replaced withanother amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart, including basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Preferably, no more than one to five conservative amino acidsubstitutions are made within the D2E7 VL and/or VH CDR3 domains. Morepreferably, no more than one to three conservative amino acidsubstitutions are made within the D2E7 VL and/or VH CDR3 domains.Additionally, conservative amino acid substitutions should not be madeat amino acid positions critical for binding to hTNFα. Positions 2 and 5of the D2E7 VL CDR3 and positions 1 and 7 of the D2E7 VH CDR3 appear tobe critical for interaction with hTNFα and thus, conservative amino acidsubstitutions preferably are not made at these positions (although analanine substitution at position 5 of the D2E7 VL CDR3 is acceptable, asdescribed above) (see U.S. Pat. No. 6,090,382).

Accordingly, in another embodiment, the invention provides methods oftreating disorders in which the administration of an anti-TNFα antibodyis beneficial by the biweekly, subcutaneous administration of anisolated human antibody, or antigen-binding portion thereof. Theantibody or antigen-binding portion thereof preferably contains thefollowing characteristics:

a) dissociates from human TNFα with a K_(off) rate constant of 1×10⁻³s⁻¹ or less, as determined by surface plasmon resonance;

b) has a light chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alaninesubstitution at position 1, 4, 5, 7 or 8 or by one to five conservativeamino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9;

c) has a heavy chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alaninesubstitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to fiveconservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9,10, 11 and/or 12.

More preferably, the antibody, or antigen-binding portion thereof,dissociates from human TNFα with a K_(off) of 5×10⁻⁴ s⁻¹ or less. Evenmore preferably, the antibody, or antigen-binding portion thereof,dissociates from human TNFα with a K_(off) of 1×10⁻⁴ s⁻¹ or less.

In yet another embodiment, the invention provides methods of treatingdisorders in which the administration of an anti-TNFα antibody isbeneficial by the biweekly, subcutaneous administration of an isolatedhuman antibody, or an antigen-binding portion thereof. The antibody orantigen-binding portion thereof preferably contains a light chainvariable region (LCVR) having a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a singlealanine substitution at position 1, 4, 5, 7 or 8, and with a heavy chainvariable region (HCVR) having a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a singlealanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11.Preferably, the LCVR further has a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 5 (i.e., the D2E7 VL CDR2) and the HCVR furtherhas a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6(i.e., the D2E7 VH CDR2). Even more preferably, the LCVR further hasCDR1 domain comprising the amino acid sequence of SEQ ID NO: 7 (i.e.,the D2E7 VL CDR1) and the HCVR has a CDR1 domain comprising the aminoacid sequence of SEQ ID NO: 8 (i.e., the D2E7 VH CDR1). The frameworkregions for VL preferably are from the V_(κ)I human germline family,more preferably from the A20 human germline Vk gene and most preferablyfrom the D2E7 VL framework sequences shown in FIGS. 1A and 1B of U.S.Pat. No. 6,090,382. The framework regions for VH preferably are from theV_(H)3 human germline family, more preferably from the DP-31 humangermline VH gene and most preferably from the D2E7 VH frameworksequences shown in FIGS. 2A and 2B U.S. Pat. No. 6,090,382.

In still another embodiment, the invention provides methods of treatingdisorders in which the administration of an anti-TNFα antibody isbeneficial by the biweekly, subcutaneous administration of an isolatedhuman antibody, or an antigen binding portion thereof. The antibody orantigen-binding portion thereof preferably contains a light chainvariable region (LCVR) comprising the amino acid sequence of SEQ ID NO:1 (i.e., the D2E7 VL) and a heavy chain variable region (HCVR)comprising the amino acid sequence of SEQ ID NO: 2 (i.e., the D2E7 VH).In certain embodiments, the antibody comprises a heavy chain constantregion, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constantregion. Preferably, the heavy chain constant region is an IgG heavychain constant region or an IgG4 heavy chain constant region.Furthermore, the antibody can comprise a light chain constant region,either a kappa light chain constant region or a lambda light chainconstant region. Preferably, the antibody comprises a kappa light chainconstant region. Alternatively, the antibody portion can be, forexample, a Fab fragment or a single chain Fv fragment.

In still other embodiments, the invention provides methods of treatingdisorders in which the administration of an anti-TNFα antibody isbeneficial by the biweekly, subcutaneous administration of an isolatedhuman antibody, or an antigen-binding portions thereof. The antibody orantigen-binding portion thereof preferably contains D2E7-related VL andVH CDR3 domains, for example, antibodies, or antigen-binding portionsthereof, with a light chain variable region (LCVR) having a CDR3 domaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 or with a heavy chainvariable region (HCVR) having a CDR3 domain comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO:27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ IDNO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35.

An antibody or antibody portion of the invention can be derivatized orlinked to another functional molecule (e.g., another peptide orprotein). Accordingly, the antibodies and antibody portions of theinvention are intended to include derivatized and otherwise modifiedforms of the human anti-hTNFα antibodies described herein, includingimmunoadhesion molecules. For example, an antibody or antibody portionof the invention can be functionally linked (by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody (e.g., a bispecificantibody or a diabody), a detectable agent, a cytotoxic agent, apharmaceutical agent, and/or a protein or peptide that can mediateassociate of the antibody or antibody portion with another molecule(such as a streptavidin core region or a polyhistidine tag).

One type of derivatized antibody is produced by crosslinking two or moreantibodies (of the same type or of different types, e.g., to createbispecific antibodies). Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Ill.

Useful detectable agents with which an antibody or antibody portion ofthe invention may be derivatized include fluorescent compounds.Exemplary fluorescent detectable agents include fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonylchloride, phycoerythrin and the like. An antibody may also bederivatized with detectable enzymes, such as alkaline phosphatase,horseradish peroxidase, glucose oxidase and the like. When an antibodyis derivatized with a detectable enzyme, it is detected by addingadditional reagents that the enzyme uses to produce a detectablereaction product. For example, when the detectable agent horseradishperoxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be derivatized with biotin, anddetected through indirect measurement of avidin or streptavidin binding.

II. Expression of Antibodies

An antibody, or antibody portion, of the invention can be prepared byrecombinant expression of immunoglobulin light and heavy chain genes ina host cell. To express an antibody recombinantly, a host cell istransfected with one or more recombinant expression vectors carrying DNAfragments encoding the immunoglobulin light and heavy chains of theantibody such that the light and heavy chains are expressed in the hostcell and, preferably, secreted into the medium in which the host cellsare cultured, from which medium the antibodies can be recovered.Standard recombinant DNA methodologies are used to obtain antibody heavyand light chain genes, incorporate these genes into recombinantexpression vectors and introduce the vectors into host cells, such asthose described in Sambrook, Fritsch and Maniatis (eds), MolecularCloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,(1989), Ausubel, F. M. et al. (eds.) Current Protocols in MolecularBiology, Greene Publishing Associates, (1989) and in U.S. Pat. No.4,816,397 by Boss et al.

To express D2E7 or a D2E7-related antibody, DNA fragments encoding thelight and heavy chain variable regions are first obtained. These DNAscan be obtained by amplification and modification of germline light andheavy chain variable sequences using the polymerase chain reaction(PCR). Germline DNA sequences for human heavy and light chain variableregion genes are known in the art (see e.g., the “Vbase” human germlinesequence database; see also Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242; Tomlinson, I.M., et al. (1992) “The Repertoire of Human Germline V_(H) SequencesReveals about Fifty Groups of V_(H) Segments with DifferentHypervariable Loops” J. Mol. Biol. 227:776-798; and Cox, J. P. L. et al.(1994) “A Directory of Human Germ-line V₇₈ Segments Reveals a StrongBias in their Usage” Eur. J. Immunol. 24:827-836; the contents of eachof which are expressly incorporated herein by reference). To obtain aDNA fragment encoding the heavy chain variable region of D2E7, or aD2E7-related antibody, a member of the V_(H)3 family of human germlineVH genes is amplified by standard PCR. Most preferably, the DP-31 VHgermline sequence is amplified. To obtain a DNA fragment encoding thelight chain variable region of D2E7, or a D2E7-related antibody, amember of the V_(κ)I family of human germline VL genes is amplified bystandard PCR. Most preferably, the A20 VL germline sequence isamplified. PCR primers suitable for use in amplifying the DP-31 germlineVH and A20 germline VL sequences can be designed based on the nucleotidesequences disclosed in the references cited supra, using standardmethods.

Once the germline VH and VL fragments are obtained, these sequences canbe mutated to encode the D2E7 or D2E7-related amino acid sequencesdisclosed herein. The amino acid sequences encoded by the germline VHand VL DNA sequences are first compared to the D2E7 or D2E7-related VHand VL amino acid sequences to identify amino acid residues in the D2E7or D2E7-related sequence that differ from germline. Then, theappropriate nucleotides of the germline DNA sequences are mutated suchthat the mutated germline sequence encodes the D2E7 or D2E7-relatedamino acid sequence, using the genetic code to determine whichnucleotide changes should be made. Mutagenesis of the germline sequencesis carried out by standard methods, such as PCR-mediated mutagenesis (inwhich the mutated nucleotides are incorporated into the PCR primers suchthat the PCR product contains the mutations) or site-directedmutagenesis.

Once DNA fragments encoding D2E7 or D2E7-related VH and VL segments areobtained (by amplification and mutagenesis of germline VH and VL genes,as described above), these DNA fragments can be further manipulated bystandard recombinant DNA techniques, for example to convert the variableregion genes to full-length antibody chain genes, to Fab fragment genesor to a scFv gene. In these manipulations, a VL- or VH-encoding DNAfragment is operatively linked to another DNA fragment encoding anotherprotein, such as an antibody constant region or a flexible linker. Theterm “operatively linked”, as used in this context, is intended to meanthat the two DNA fragments are joined such that the amino acid sequencesencoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the VH region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (CH1, CH2and CH3). The sequences of human heavy chain constant region genes areknown in the art (see e.g., Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably isan IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene,the VH-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat, E. A., etal. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region, but most preferably is a kappaconstant region.

To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the VH and VLsequences can be expressed as a contiguous single-chain protein, withthe VL and VH regions joined by the flexible linker (see e.g., Bird etal. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554).

To express the antibodies, or antibody portions of the invention, DNAsencoding partial or full-length light and heavy chains, obtained asdescribed above, are inserted into expression vectors such that thegenes are operatively linked to transcriptional and translationalcontrol sequences. In this context, the term “operatively linked” isintended to mean that an antibody gene is ligated into a vector suchthat transcriptional and translational control sequences within thevector serve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into separate vector or, more typically, both genesare inserted into the same expression vector. The antibody genes areinserted into the expression vector by standard methods (e.g., ligationof complementary restriction sites on the antibody gene fragment andvector, or blunt end ligation if no restriction sites are present).Prior to insertion of the D2E7 or D2E7-related light or heavy chainsequences, the expression vector may already carry antibody constantregion sequences. For example, one approach to converting the D2E7 orD2E7-related VH and VL sequences to full-length antibody genes is toinsert them into expression vectors already encoding heavy chainconstant and light chain constant regions, respectively, such that theVH segment is operatively linked to the CH segment(s) within the vectorand the VL segment is operatively linked to the CL segment within thevector. Additionally or alternatively, the recombinant expression vectorcan encode a signal peptide that facilitates secretion of the antibodychain from a host cell. The antibody chain gene can be cloned into thevector such that the signal peptide is linked in-frame to the aminoterminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to includes promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. For furtherdescription of viral regulatory elements, and sequences thereof, seee.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 byBell et al. and U.S. Pat. No. 4,968,615 by Schaffner et al.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody. Prokaryoticexpression of antibody genes has been reported to be ineffective forproduction of high yields of active antibody (Boss, M. A. and Wood, C.R. (1985) Immunology Today 6:12-13).

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding antibody genes are introducedinto mammalian host cells, the antibodies are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe antibody in the host cells or, more preferably, secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods.

Host cells can also be used to produce portions of intact antibodies,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure are within the scope of the presentinvention. For example, it may be desirable to transfect a host cellwith DNA encoding either the light chain or the heavy chain (but notboth) of an antibody of this invention. Recombinant DNA technology mayalso be used to remove some or all of the DNA encoding either or both ofthe light and heavy chains that is not necessary for binding to hTNFα.The molecules expressed from such truncated DNA molecules are alsoencompassed by the antibodies of the invention. In addition,bifunctional antibodies may be produced in which one heavy and one lightchain are an antibody of the invention and the other heavy and lightchain are specific for an antigen other than hTNFα by crosslinking anantibody of the invention to a second antibody by standard chemicalcrosslinking methods.

In a preferred system for recombinant expression of an antibody, orantigen-binding portion thereof, of the invention, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to CMV enhancer/AdMLP promoter regulatory elements to drive highlevels of transcription of the genes. The recombinant expression vectoralso carries a DHFR gene, which allows for selection of CHO cells thathave been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells areculture to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells and recover the antibody from the culture medium.

III. Selection of Recombinant Human Antibodies

Recombinant human antibodies of the invention in addition to D2E7 or anantigen binding portion thereof, or D2E7-related antibodies disclosedherein can be isolated by screening of a recombinant combinatorialantibody library, preferably a scFv phage display library, preparedusing human VL and VH cDNAs prepared from mRNA derived from humanlymphocytes. Methodologies for preparing and screening such librariesare known in the art. In addition to commercially available kits forgenerating phage display libraries (e.g., the Pharmacia RecombinantPhage Antibody System, catalog no. 27-9400-01; and the StratageneSurfZAP™ phage display kit, catalog no. 240612), examples of methods andreagents particularly amenable for use in generating and screeningantibody display libraries can be found in, for example, Ladner et al.U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No. WO 92/18619;Dower et al. PCT Publication No. WO 91/17271; Winter et al. PCTPublication No. WO 92/20791; Markland et al. PCT Publication No. WO92/15679; Breitling et al. PCT Publication No. WO 93/01288; McCaffertyet al. PCT Publication No. WO 92/01047; Garrard et al. PCT PublicationNo. WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay etal. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffithset al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992) PNAS 89:3576-3580; Garrard et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; andBarbas et al. (1991) PNAS 88:7978-7982.

In a preferred embodiment, to isolate human antibodies with highaffinity and a low off rate constant for hTNFα, a murine anti-hTNFαantibody having high affinity and a low off rate constant for hTNFα(e.g., MAK 195, the hybridoma for which has deposit number ECACC 87050801) is first used to select human heavy and light chain sequenceshaving similar binding activity toward hTNFα, using the epitopeimprinting methods described in Hoogenboom et al., PCT Publication No.WO 93/06213. The antibody libraries used in this method are preferablyscFv libraries prepared and screened as described in McCafferty et al.,PCT Publication No. WO 92/01047, McCafferty et al., Nature (1990)348:552-554; and Griffiths et al., (1993) EMBO J 12:725-734. The scFvantibody libraries preferably are screened using recombinant human TNFαas the antigen.

Once initial human VL and VH segments are selected, “mix and match”experiments, in which different pairs of the initially selected VL andVH segments are screened for hTNFα binding, are performed to selectpreferred VL/VH pair combinations. Additionally, to further improve theaffinity and/or lower the off rate constant for hTNFα binding, the VLand VH segments of the preferred VL/VH pair(s) can be randomly mutated,preferably within the CDR3 region of VH and/or VL, in a processanalogous to the in vivo somatic mutation process responsible foraffinity maturation of antibodies during a natural immune response. Thisin vitro affinity maturation can be accomplished by amplifying VH and VLregions using PCR primers complimentary to the VH CDR3 or VL CDR3,respectively, which primers have been “spiked” with a random mixture ofthe four nucleotide bases at certain positions such that the resultantPCR products encode VH and VL segments into which random mutations havebeen introduced into the VH and/or VL CDR3 regions. These randomlymutated VH and VL segments can be rescreened for binding to hTNFα andsequences that exhibit high affinity and a low off rate for hTNFαbinding can be selected.

Following screening and isolation of an anti-hTNFα antibody of theinvention from a recombinant immunoglobulin display library, nucleicacid encoding the selected antibody can be recovered from the displaypackage (e.g., from the phage genome) and subcloned into otherexpression vectors by standard recombinant DNA techniques. If desired,the nucleic acid can be further manipulated to create other antibodyforms of the invention (e.g., linked to nucleic acid encoding additionalimmunoglobulin domains, such as additional constant regions). To expressa recombinant human antibody isolated by screening of a combinatoriallibrary, the DNA encoding the antibody is cloned into a recombinantexpression vector and introduced into a mammalian host cells, asdescribed in further detail in Section II above.

IV. Pharmaceutical Compositions and Pharmaceutical Administration

The antibodies and antibody-portions of the invention can beincorporated into pharmaceutical compositions suitable foradministration to a subject for the methods described herein, e.g.,biweekly, subcutaneous dosing. Typically, the pharmaceutical compositioncomprises an antibody (or antibody portion) of the invention and/ormethotrexate and a pharmaceutically acceptable carrier. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible and are suitable for administration to asubject for the methods described herein. Examples of pharmaceuticallyacceptable carriers include one or more of water, saline, phosphatebuffered saline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Pharmaceuticallyacceptable carriers may further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of the antibodyor antibody portion.

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans with other antibodies. Thepreferred mode of administration is parenteral (e.g., intravenous,subcutaneous, intraperitoneal, intramuscular). In a preferredembodiment, the antibody is administered by intravenous infusion orinjection. In another preferred embodiment, the antibody is administeredby intramuscular injection. In a particularly preferred embodiment, theantibody is administered by subcutaneous injection (e.g., a biweekly,subcutaneous injection).

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antibody or antibody portion) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying that yields apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof. The proper fluidityof a solution can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prolongedabsorption of injectable compositions can be brought about by includingin the composition an agent that delays absorption, for example,monostearate salts and gelatin.

The antibodies and antibody-portions of the present invention can beadministered by a variety of methods known in the art, although for manytherapeutic applications, the preferred route/mode of administration issubcutaneous injection. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results. In certain embodiments, the active compound may beprepared with a carrier that will protect the compound against rapidrelease, such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyethylene glycol (PEG), polyanhydrides, polyglycolicacid, collagen, polyorthoesters, and polylactic acid. Many methods forthe preparation of such formulations are patented or generally known tothose skilled in the art. See, e.g., Sustained and Controlled ReleaseDrug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., NewYork, 1978.

In certain embodiments, an antibody or antibody portion of the inventionmay be orally administered, for example, with an inert diluent or anassimilable edible carrier. The compound (and other ingredients, ifdesired) may also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the compounds may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. To administer a compound of the invention by other thanparenteral administration, it may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, an antibody or antibody portion ofthe invention is coformulated with and/or coadministered with one ormore additional therapeutic agents. For example, an anti-hTNFα antibodyor antibody portion of the invention may be coformulated and/orcoadministered with methotrexate, one or more additional antibodies thatbind other targets (e.g., antibodies that bind other cytokines or thatbind cell surface molecules), one or more cytokines, soluble TNFαreceptor (see e.g., PCT Publication No. WO 94/06476) and/or one or morechemical agents that inhibit hTNFα production or activity (such ascyclohexane-ylidene derivatives as described in PCT Publication No. WO93/19751). Furthermore, one or more antibodies of the invention may beused in combination with two or more of the foregoing therapeuticagents. Such combination therapies may advantageously utilize lowerdosages of the administered therapeutic agents, thus avoiding possibletoxicities or complications associated with the various monotherapies.The use of the antibodies, or antibody portions, of the invention incombination with other therapeutic agents is discussed further insubsection IV.

Non-limiting examples of therapeutic agents for rheumatoid arthritiswith which an antibody, or antibody portion, of the invention can becombined include the following: non-steroidal anti-inflammatory drug(s)(NSAIDs); cytokine suppressive anti-inflammatory drug(s) (CSAIDs);CDP-571/BAY-10-3356 (humanized anti-TNFα antibody; Celltech/Bayer); cA2(chimeric anti-TNFα antibody; Centocor); 75 kdTNFR-IgG (75 kD TNFαreceptor-IgG fusion protein; Immunex; see e.g., Arthritis & Rheumatism(1994) Vol. 37, S295; J. Invest. Med. (1996) Vol. 44, 235A); 55kdTNFR-IgG (55 kD TNFα receptor-IgG fusion protein; Hoffmann-LaRoche);IDEC-CE9.1/SB 210396 (non-depleting primatized anti-CD4 antibody;IDEC/SmithKline; see e.g., Arthritis & Rheumatism (1995) Vol. 38, S185);DAB 486-IL-2 and/or DAB 389-IL-2 (IL-2 fusion proteins; Seragen; seee.g., Arthritis & Rheumatism (1993) Vol. 36, 1223); Anti-Tac (humanizedanti-IL-2Ra; Protein Design Labs/Roche); IL-4 (anti-inflammatorycytokine; DNAX/Schering); IL-10 (SCH 52000; recombinant IL-10,anti-inflammatory cytokine; DNAX/Schering); IL-4; IL-10 and/or IL-4agonists (e.g., agonist antibodies); IL-RA (IL-1 receptor antagonist;Synergen/Amgen); TNF-bp/s-TNFR (soluble TNFα binding protein; see e.g.,Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S284; Amer.J. Physiol.—Heart and Circulatory Physiology (1995) Vol. 268, pp.37-42); R973401 (phosphodiesterase Type IV inhibitor; see e.g.,Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S282); MK-966(COX-2 Inhibitor; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), S81); Iloprost (see e.g., Arthritis & Rheumatism (1996)Vol. 39, No. 9 (supplement), S82); methotrexate; thalidomide (see e.g.,Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S282) andthalidomide-related drugs (e.g., Celgen); leflunomide (anti-inflammatoryand cytokine inhibitor; see e.g., Arthritis & Rheumatism (1996) Vol. 39,No. 9 (supplement), S131; Inflammation Research (1996) Vol. 45, pp.103-107); tranexamic acid (inhibitor of plasminogen activation; seee.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S284);T-614 (cytokine inhibitor; see e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (supplement), S282); prostaglandin E1 (see e.g., Arthritis &Rheumatism (1996) Vol. 39, No. 9 (supplement), S282); Tenidap(non-steroidal anti-inflammatory drug; see e.g., Arthritis & Rheumatism(1996) Vol. 39, No. 9 (supplement), S280); Naproxen (non-steroidalanti-inflammatory drug; see e.g., Neuro Report (1996) Vol. 7, pp.1209-1213); Meloxicam (non-steroidal anti-inflammatory drug); Ibuprofen(non-steroidal anti-inflammatory drug); Piroxicam (non-steroidalanti-inflammatory drug); Diclofenac (non-steroidal anti-inflammatorydrug); Indomethacin (non-steroidal anti-inflammatory drug);Sulfasalazine (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), S281); Azathioprine (see e.g., Arthritis & Rheumatism(1996) Vol. 39, No. 9 (supplement), S281); ICE inhibitor (inhibitor ofthe enzyme interleukin-1β converting enzyme); zap-70 and/or Ickinhibitor (inhibitor of the tyrosine kinase zap-70 or Ick); VEGFinhibitor and/or VEGF-R inhibitor (inhibitors of vascular endothelialcell growth factor or vascular endothelial cell growth factor receptor;inhibitors of angiogenesis); corticosteroid anti-inflammatory drugs(e.g., SB203580); TNF-convertase inhibitors; anti-IL-12 antibodies;interleukin-11 (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), S296); interleukin-13 (see e.g., Arthritis & Rheumatism(1996) Vol. 39, No. 9 (supplement), S308); interleukin-17 inhibitors(see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement),S120); gold; penicillamine; chloroquine; hydroxychloroquine;chlorambucil; cyclophosphamide; cyclosporine; total lymphoidirradiation; anti-thymocyte globulin; anti-CD4 antibodies; CD5-toxins;orally-administered peptides and collagen; lobenzarit disodium; CytokineRegulating Agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals,Inc.); ICAM-1 antisense phosphorothioate oligodeoxynucleotides (ISIS2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10;T Cell Sciences, Inc.); prednisone; orgotein; glycosaminoglycanpolysulphate; minocycline; anti-IL2R antibodies; marine and botanicallipids (fish and plant seed fatty acids; see e.g., DeLuca et al. (1995)Rheum. Dis. Clin. North Am. 21:759-777); auranofin; phenylbutazone;meclofenamic acid; flufenamic acid; intravenous immune globulin;zileuton; mycophenolic acid (RS-61443); tacrolimus (FK-506); sirolimus(rapamycin); amiprilose (therafectin); cladribine(2-chlorodeoxyadenosine); and azaribine.

Non-limiting examples of therapeutic agents for inflammatory boweldisease with which an antibody, or antibody portion, of the inventioncan be combined include the following: budenoside; epidermal growthfactor; corticosteroids; cyclosporin, sulfasalazine; aminosalicylates;6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors;mesalamine; olsalazine; balsalazide; antioxidants; thromboxaneinhibitors; IL-1 receptor antagonists; anti-IL-1β monoclonal antibodies;anti-IL-6 monoclonal antibodies; growth factors; elastase inhibitors;pyridinyl-imidazole compounds; CDP-571/BAY-10-3356 (humanized anti-TNFαantibody; Celltech/Bayer); cA2 (chimeric anti-TNFα antibody; Centocor);75 kdTNFR-IgG (75 kD TNFα receptor-IgG fusion protein; Immunex; seee.g., Arthritis & Rheumatism (1994) Vol. 37, S295; J. Invest. Med.(1996) Vol. 44, 235A); 55 kdTNFR-IgG (55 kD TNFα receptor-IgG fusionprotein; Hoffmann-LaRoche); interleukin-10 (SCH 52000; Schering Plough);IL-4; IL-10 and/or IL-4 agonists (e.g., agonist antibodies);interleukin-11; glucuronide- or dextran-conjugated prodrugs ofprednisolone, dexamethasone or budesonide; ICAM-1 antisensephosphorothioate oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals,Inc.); soluble complement receptor 1 (TP10; T Cell Sciences, Inc.);slow-release mesalazine; methotrexate; antagonists of PlateletActivating Factor (PAF); ciprofloxacin; and lignocaine.

Nonlimiting examples of therapeutic agents for multiple sclerosis withwhich an antibody, or antibody portion, of the invention can be combinedinclude the following: corticosteroids; prednisolone;methylprednisolone; azathioprine; cyclophosphamide; cyclosporine;methotrexate; 4-aminopyridine; tizanidine; interferon-β1a (Avonex™;Biogen); interferon-β1b (Betaseron™; Chiron/Berlex); Copolymer 1 (Cop-1;Copaxone™; Teva Pharmaceutical Industries, Inc.); hyperbaric oxygen;intravenous immunoglobulin; clabribine; CDP-571/BAY-10-3356 (humanizedanti-TNFα antibody; Celltech/Bayer); cA2 (chimeric anti-TNFα antibody;Centocor); 75 kdTNFR-IgG (75 kD TNFα receptor-IgG fusion protein;Immunex; see e.g., Arthritis & Rheumatism (1994) Vol. 37, S295; J.Invest. Med. (1996) Vol. 44, 235A); 55 kdTNFR-IgG (55 kD TNFαreceptor-IgG fusion protein; Hoffmann-LaRoche); IL-10; IL-4; and IL-10and/or IL-4 agonists (e.g., agonist antibodies).

Nonlimiting examples of therapeutic agents for sepsis with which anantibody, or antibody portion, of the invention can be combined includethe following: hypertonic saline solutions; antibiotics; intravenousgamma globulin; continuous hemofiltration; carbapenems (e.g.,meropenem); antagonists of cytokines such as TNFα, IL-1β, IL-6 and/orIL-8; CDP-571/BAY-10-3356 (humanized anti-TNFα antibody;Celltech/Bayer); cA2 (chimeric anti-TNFα antibody; Centocor); 75kdTNFR-IgG (75 kD TNFα receptor-IgG fusion protein; Immunex; see e.g.,Arthritis & Rheumatism (1994) Vol. 37, S295; J. Invest. Med. (1996) Vol.44, 235A); 55 kdTNFR-IgG (55 kD TNFα receptor-IgG fusion protein;Hoffmann-LaRoche); Cytokine Regulating Agents (CRAs) HP228 and HP466(Houghten Pharmaceuticals, Inc.); SK&F 107647 (low molecular peptide;SmithKline Beecham); tetravalent guanylhydrazone CNI-1493 (PicowerInstitute); Tissue Factor Pathway Inhibitor (TFPI; Chiron); PHP(chemically modified hemoglobin; APEX Bioscience); iron chelators andchelates, including diethylenetriamine pentaacetic acid-iron (III)complex (DTPA iron (III); Molichem Medicines); lisofylline (syntheticsmall molecule methylxanthine; Cell Therapeutics, Inc.); PGG-Glucan(aqeuous soluble β1,3glucan; Alpha-Beta Technology); apolipoprotein A-1reconstituted with lipids; chiral hydroxamic acids (syntheticantibacterials that inhibit lipid A biosynthesis); anti-endotoxinantibodies; E5531 (synthetic lipid A antagonist; Eisai America, Inc.);rBPI₂₁ (recombinant N-terminal fragment of humanBactericidal/Permeability-Increasing Protein); and SyntheticAnti-Endotoxin Peptides (SAEP; BiosYnth Research Laboratories);

Nonlimiting examples of therapeutic agents for adult respiratorydistress syndrome (ARDS) with which an antibody, or antibody portion, ofthe invention can be combined include the following: anti-IL-8antibodies; surfactant replacement therapy; CDP-571/BAY-10-3356(humanized anti-TNFα antibody; Celltech/Bayer); cA2 (chimeric anti-TNFαantibody; Centocor); 75 kdTNFR-IgG (75 kD TNFα receptor-IgG fusionprotein; Immunex; see e.g., Arthritis & Rheumatism (1994) Vol. 37, S295;J. Invest. Med. (1996) Vol. 44, 235A); and 55 kdTNFR-IgG (55 kD TNFαreceptor-IgG fusion protein; Hoffmann-LaRoche).

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antibody portion of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the antibodyor antibody portion may vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of theantibody or antibody portion to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or antibody portion ofthe invention is 10-100 mg, more preferably 20-80 mg and most preferablyabout 40 mg. It is to be noted that dosage values may vary with the typeand severity of the condition to be alleviated. It is to be furtherunderstood that for any particular subject, specific dosage regimensshould be adjusted over time according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the compositions, and that dosage ranges set forthherein are exemplary only and are not intended to limit the scope orpractice of the claimed composition.

V. Uses of the Antibodies of the Invention

Given their ability to bind to hTNFα, the anti-hTNFα antibodies, orportions thereof, of the invention can be used to detect hTNFα (e.g., ina biological sample, such as serum or plasma), using a conventionalimmunoassay, such as an enzyme linked immunosorbent assays (ELISA), anradioimmunoassay (RIA) or tissue immunohistochemistry. The inventionprovides a method for detecting hTNFα in a biological sample comprisingcontacting a biological sample with an antibody, or antibody portion, ofthe invention and detecting either the antibody (or antibody portion)bound to hTNFα or unbound antibody (or antibody portion), to therebydetect hTNFα in the biological sample. The antibody is directly orindirectly labeled with a detectable substance to facilitate detectionof the bound or unbound antibody. Suitable detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials and radioactive materials. Examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase, β-galactosidase,or acetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; and examples of suitable radioactive material include ¹²⁵I,¹³¹I, ³⁵S or ³H.

Alternative to labeling the antibody, hTNFα can be assayed in biologicalfluids by a competition immunoassay utilizing rhTNFα standards labeledwith a detectable substance and an unlabeled anti-hTNFα antibody. Inthis assay, the biological sample, the labeled rhTNFα standards and theanti-hTNFα antibody are combined and the amount of labeled rhTNFαstandard bound to the unlabeled antibody is determined. The amount ofhTNFα in the biological sample is inversely proportional to the amountof labeled hTNFα standard bound to the anti-hTNFα antibody.

A D2E7 antibody of the invention can also be used to detect TNFαs fromspecies other than humans, in particular TNFαs from primates (e.g.,chimpanzee, baboon, marmoset, cynomolgus and rhesus), pig and mouse,since D2E7 can bind to each of these TNFαs.

The antibodies and antibody portions of the invention are capable ofneutralizing hTNFα activity both in vitro and in vivo (see U.S. Pat. No.6,090,382). Moreover, at least some of the antibodies of the invention,such as D2E7, can neutralize hTNFα activity from other species.Accordingly, the antibodies and antibody portions of the invention canbe used to inhibit hTNFα activity, e.g., in a cell culture containinghTNFα, in human subjects or in other mammalian subjects having TNFαswith which an antibody of the invention cross-reacts (e.g. chimpanzee,baboon, marmoset, cynomolgus and rhesus, pig or mouse). In oneembodiment, the invention provides a method for inhibiting TNFα activitycomprising contacting TNFα with an antibody or antibody portion of theinvention such that TNFα activity is inhibited. Preferably, the TNFα ishuman TNFα. For example, in a cell culture containing, or suspected ofcontaining TNFα, an antibody or antibody portion of the invention can beadded to the culture medium to inhibit hTNFα activity in the culture.

In a preferred embodiment, the invention provides methods of treatingdisorders in which the administration of an anti-TNFα antibody isbeneficial, comprising subcutaneously administering to the subjectbiweekly an antibody or antibody portion of the invention such that thedisorder is treated. In a particularly preferred embodiment, theantibody is administered subcutaneously on a biweekly schedule. Inanother particularly preferred embodiment, the antibody is administeredsubcutaneously before, during or after administration of methotrexate.Preferably, the subject is a human subject. Alternatively, the subjectcan be a mammal expressing a TNFα with which an antibody of theinvention cross-reacts. Still further the subject can be a mammal intowhich has been introduced hTNFα(e.g., by administration of hTNFα or byexpression of an hTNFα transgene). An antibody of the invention can beadministered to a human subject for therapeutic purposes (discussedfurther below). Moreover, an antibody of the invention can beadministered to a non-human mammal expressing a TNFα with which theantibody cross-reacts (e.g., a primate, pig or mouse) for veterinarypurposes or as an animal model of human disease. Regarding the latter,such animal models may be useful for evaluating the therapeutic efficacyof antibodies of the invention (e.g., testing of dosages and timecourses of administration).

As used herein, the term “a disorder in which the administration of ananti-TNFα antibody is beneficial” is intended to include diseases andother disorders in which the presence of TNFα in a subject sufferingfrom the disorder has been shown to be or is suspected of being eitherresponsible for the pathophysiology of the disorder or a factor thatcontributes to a worsening of the disorder, or where it has been shownthat another anti-TNFα antibody or a biologically active portion thereofhas been successfully used to treat the disease. Accordingly, a disorderin which TNFα activity is detrimental is a disorder in which inhibitionof TNFα activity is expected to alleviate the symptoms and/orprogression of the disorder. Such disorders may be evidenced, forexample, by an increase in the concentration of TNFα in a biologicalfluid of a subject suffering from the disorder (e.g., an increase in theconcentration of TNFα in serum, plasma, synovial fluid, etc. of thesubject), which can be detected, for example, using an anti-TNFαantibody as described above. There are numerous examples of disorders inwhich TNFα activity is detrimental. The use of the antibodies andantibody portions of the invention in the treatment of specificdisorders is discussed further below:

A. Sepsis

Tumor necrosis factor has an established role in the pathophysiology ofsepsis, with biological effects that include hypotension, myocardialsuppression, vascular leakage syndrome, organ necrosis, stimulation ofthe release of toxic secondary mediators and activation of the clottingcascade (see e.g., Tracey, K. J. and Cerami, A. (1994) Annu. Rev. Med.45:491-503; Russell, D and Thompson, R. C. (1993) Curr. Opin. Biotech.4:714-721). Accordingly, the human antibodies, and antibody portions, ofthe invention can be used to treat sepsis in any of its clinicalsettings, including septic shock, endotoxic shock, gram negative sepsisand toxic shock syndrome.

Furthermore, to treat sepsis, an anti-hTNFα antibody, or antibodyportion, of the invention can be coadministered with one or moreadditional therapeutic agents that may further alleviate sepsis, such asan interleukin-1 inhibitor (such as those described in PCT PublicationNos. WO 92/16221 and WO 92/17583), the cytokine interleukin-6 (see e.g.,PCT Publication No. WO 93/11793) or an antagonist of platelet activatingfactor (see e.g., European Patent Application Publication No. EP 374510).

Additionally, in a preferred embodiment, an anti-TNFα antibody orantibody portion of the invention is administered to a human subjectwithin a subgroup of sepsis patients having a serum or plasmaconcentration of IL-6 above 500 pg/ml, and more preferably 1000 pg/ml,at the time of treatment (see PCT Publication No. WO 95/20978 by Daum,L., et al.).

B. Autoimmune Diseases

Tumor necrosis factor has been implicated in playing a role in thepathophysiology of a variety of autoimmune diseases. For example, TNFαhas been implicated in activating tissue inflammation and causing jointdestruction in rheumatoid arthritis (see e.g., Tracey and Cerami, supra;Arend, W. P. and Dayer, J- M. (1995) Arth. Rheum. 38:151-160; Fava, R.A., et al. (1993) Clin. Exp. Immunol. 94:261-266). TNFα also has beenimplicated in promoting the death of islet cells and in mediatinginsulin resistance in diabetes (see e.g., Tracey and Cerami, supra; PCTPublication No. WO 94/08609). TNFα also has been implicated in mediatingcytotoxicity to oligodendrocytes and induction of inflammatory plaquesin multiple sclerosis (see e.g., Tracey and Cerami, supra). Chimeric andhumanized murine anti-hTNFα antibodies have undergone clinical testingfor treatment of rheumatoid arthritis (see e.g., Elliott, M. J., et al.(1994) Lancet 344:1125-1127; Elliot, M. J., et al. (1994) Lancet344:1105-1110; Rankin, E. C., et al. (1995) Br. J. Rheumatol.34:334-342).

The human antibodies, and antibody portions of the invention can be usedto treat autoimmune diseases, in particular those associated withinflammation, including rheumatoid arthritis, rheumatoid spondylitis,osteoarthritis and gouty arthritis, allergy, multiple sclerosis,autoimmune diabetes, autoimmune uveitis and nephrotic syndrome.Typically, the antibody, or antibody portion, is administeredsystemically, although for certain disorders, local administration ofthe antibody or antibody portion at a site of inflammation may bebeneficial (e.g., local administration in the joints in rheumatoidarthritis or topical application to diabetic ulcers, alone or incombination with a cyclohexane-ylidene derivative as described in PCTPublication No. WO 93/19751).

C. Infectious Diseases

Tumor necrosis factor has been implicated in mediating biologicaleffects observed in a variety of infectious diseases. For example, TNFαhas been implicated in mediating brain inflammation and capillarythrombosis and infarction in malaria (see e.g., Tracey and Cerami,supra). TNFα also has been implicated in mediating brain inflammation,inducing breakdown of the blood-brain barrier, triggering septic shocksyndrome and activating venous infarction in meningitis (see e.g.,Tracey and Cerami, supra). TNFα also has been implicated in inducingcachexia, stimulating viral proliferation and mediating central nervoussystem injury in acquired immune deficiency syndrome (AIDS) (see e.g.,Tracey and Cerami, supra). Accordingly, the antibodies, and antibodyportions, of the invention, can be used in the treatment of infectiousdiseases, including bacterial meningitis (see e.g., European PatentApplication Publication No. EP 585 705), cerebral malaria, AIDS andAIDS-related complex (ARC) (see e.g., European Patent ApplicationPublication No. EP 230 574), as well as cytomegalovirus infectionsecondary to transplantation (see e.g., Fietze, E., et al. (1994)Transplantation 58:675-680). The antibodies, and antibody portions, ofthe invention, also can be used to alleviate symptoms associated withinfectious diseases, including fever and myalgias due to infection (suchas influenza) and cachexia secondary to infection (e.g., secondary toAIDS or ARC).

D. Transplantation

Tumor necrosis factor has been implicated as a key mediator of allograftrejection and graft versus host disease (GVHD) and in mediating anadverse reaction that has been observed when the rat antibody OKT3,directed against the T cell receptor CD3 complex, is used to inhibitrejection of renal transplants (see e.g., Tracey and Cerami, supra;Eason, J. D., et al. (1995) Transplantation 59:300-305; Suthanthiran, M.and Strom, T. B. (1994) New Engl. J. Med. 331:365-375). Accordingly, theantibodies, and antibody portions, of the invention, can be used toinhibit transplant rejection, including rejections of allografts andxenografts and to inhibit GVHD. Although the antibody or antibodyportion may be used alone, more preferably it is used in combinationwith one or more other agents that inhibit the immune response againstthe allograft or inhibit GVHD. For example, in one embodiment, anantibody or antibody portion of the invention is used in combinationwith OKT3 to inhibit OKT3-induced reactions. In another embodiment, anantibody or antibody portion of the invention is used in combinationwith one or more antibodies directed at other targets involved inregulating immune responses, such as the cell surface molecules CD25(interleukin-2 receptor-α), CD11a (LFA-1), CD54 (ICAM-1), CD4, CD45,CD28/CTLA4, CD80 (B7-1) and/or CD86 (B7-2). In yet another embodiment,an antibody or antibody portion of the invention is used in combinationwith one or more general immunosuppressive agents, such as cyclosporin Aor FK506.

E. Malignancy

Tumor necrosis factor has been implicated in inducing cachexia,stimulating tumor growth, enhancing metastatic potential and mediatingcytotoxicity in malignancies (see e.g., Tracey and Cerami, supra).Accordingly, the antibodies, and antibody portions, of the invention,can be used in the treatment of malignancies, to inhibit tumor growth ormetastasis and/or to alleviate cachexia secondary to malignancy. Theantibody, or antibody portion, may be administered systemically orlocally to the tumor site.

F. Pulmonary Disorders

Tumor necrosis factor has been implicated in the pathophysiology ofadult respiratory distress syndrome, including stimulatingleukocyte-endothelial activation, directing cytotoxicity to pneumocytesand inducing vascular leakage syndrome (see e.g., Tracey and Cerami,supra). Accordingly, the antibodies, and antibody portions, of theinvention, can be used to treat various pulmonary disorders, includingadult respiratory distress syndrome (see e.g., PCT Publication No. WO91/04054), shock lung, chronic pulmonary inflammatory disease, pulmonarysarcoidosis, pulmonary fibrosis and silicosis. The antibody, or antibodyportion, may be administered systemically or locally to the lungsurface, for example as an aerosol.

G. Intestinal Disorders

Tumor necrosis factor has been implicated in the pathophysiology ofinflammatory bowel disorders (see e.g., Tracy, K. J., et al. (1986)Science 234:470-474; Sun, X- M., et al. (1988) J. Clin. Invest.81:1328-1331; MacDonald, T. T., et al. (1990) Clin. Exp. Immunol.81:301-305). Chimeric murine anti-hTNFα antibodies have undergoneclinical testing for treatment of Crohn's disease (van Dullemen, H. M.,et al. (1995) Gastroenterology 109:129-135). The human antibodies, andantibody portions, of the invention, also can be used to treatintestinal disorders, such as idiopathic inflammatory bowel disease,which includes two syndromes, Crohn's disease and ulcerative colitis.

H. Cardiac Disorders

The antibodies, and antibody portions, of the invention, also can beused to treat various cardiac disorders, including ischemia of the heart(see e.g., European Patent Application Publication No. EP 453 898) andheart insufficiency (weakness of the heart muscle) (see e.g., PCTPublication No. WO 94/20139).

I. Others

The antibodies, and antibody portions, of the invention, also can beused to treat various other disorders in which TNFα activity isdetrimental. Examples of other diseases and disorders in which TNFαactivity has been implicated in the pathophysiology, and thus which canbe treated using an antibody, or antibody portion, of the invention,include inflammatory bone disorders and bone resorption disease (seee.g., Bertolini, D. R., et al. (1986) Nature 319:516-518; Konig, A., etal. (1988) J. Bone Miner. Res. 3:621-627; Lerner, U. H. and Ohlin, A.(1993) J. Bone Miner. Res. 8:147-155; and Shankar, G. and Stern, P. H.(1993) Bone 14:871-876), hepatitis, including alcoholic hepatitis (seee.g., McClain, C. J. and Cohen, D. A. (1989) Hepatology 9:349-351;Felver, M. E., et al. (1990) Alcohol. Clin. Exp. Res. 14:255-259; andHansen, J., et al. (1994) Hepatology 20:461-474) and viral hepatitis(Sheron, N., et al. (1991) J. Hepatol. 12:241-245; and Hussain, M. J.,et al. (1994) J. Clin. Pathol. 47:1112-1115), coagulation disturbances(see e.g., van der Poll, T., et al. (1990) N. Engl. J. Med.322:1622-1627; and van der Poll, T., et al. (1991) Prog. Clin. Biol.Res. 367:55-60), Burns (see e.g., Giroir, B. P., et al. (1994) Am. J.Physiol. 267:H118-124; and Liu, X. S., et al. (1994) Burns 20:40-44),reperfusion injury (see e.g., Scales, W. E., et al. (1994) Am. J.Physiol. 267:G1122-1127; Serrick, C., et al. (1994) Transplantation58:1158-1162; and Yao, Y. M., et al. (1995) Resuscitation 29:157-168),keloid formation (see e.g., McCauley, R. L., et al. (1992) J. Clin.Immunol. 12:300-308), scar tissue formation and pyrexia.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are hereby incorporated by reference.

Example 1 Treatment with an Anti-TNFα Antibody D2E7 Efficacy FollowingSubcutaneous Administration

In this study, twenty-four patients with active RA were treated withweekly doses of 0.5 mg/kg D2E7 (n=18) or placebo (n=6) by s.c. injectionfor three months. Patients participating in this study had a meanduration of disease of 10.1 years with a disease activity score (DAS)score of 4.87 and a mean of 3.4 DMARDs (disease modifying anti-rheumaticdrugs) prior to study entry; again reflecting considerable diseaseactivity. Responders continued open-label treatment with D2E7, whilepatients who failed to respond to the 0.5 mg/kg dose or who lost a DASresponse on the 0.5 mg/kg dose were escalated to receive 1 mg/kg by s.c.injection after week twelve of the study.

The first patients enrolled received up to sixty injections and were,therefore, sixty weeks on the study drug. The efficacy with s.c. dosingwas similar to i.v. injections. Up to 78% of patients reached a DAS andACR20 response during the first weeks of treatment. Subcutaneous D2E7 ata dose of 0.5 mg/kg/week reduced the swollen joint (SWJ) count by 54%,tender joint count (TJC) by 61% and CRP by 39% over twelve weekscompared to baseline, whereas all parameters increased in the placebogroup. After completion of the placebo-controlled period of this study,the patients continued treatment for up to fourteen months withsustained efficacy. These results indicate that subcutaneous D2E7 at adose of 0.5 mg/kg/week can, therefore, be safely self-administered withgood local tolerability.

Administration of D2E7 and Methotrexate

In this study, patients received s.c. or i.v. placebo or D2E7 at a doseof 1 mg/kg in addition to their ongoing treatment with (methotrexate)MTX. Fifty-four patients were enrolled in the study and eighteenpatients received i.v. D2E7 and s.c. placebo, eighteen patients receivedi.v. placebo and s.c. D2E7, and eighteen patients received placebo i.v.and s.c. The patients received their second dose only after they losttheir blinded response status, not earlier than four weeks after thefirst dose. Thereafter, all patients received open-label biweekly s.c.injections of D2E7.

Demographic characteristics of the study population of this studyincluded a mean duration of RA of 11.1 years, prior exposure to a meanof 3.6 DMARDs (other than MTX), and a mean DAS at study entry of 4.81.By Day twenty-nine, 72% of the i.v. D2E7 treated patients and 44% of thes.c. D2E7 treated patients had achieved a response by DAS criteria,compared to only 28% of placebo-treated patients (set forth in FIG. 5).Of the responders in this study, 28% of placebo treated patientsmaintained an ACR20 response up to day 29, compared to 72% ofi.v.-treated D2E7 patients and 67% of s.c.-treated D2E7 patients, whomaintained their responses for between one and three months.

Example 2 Total Body Dose of a Subcutaneously Administered Anti-TNFαAntibody Weekly, Subcutaneous Administration of D2E7

This study enrolled two hundred eighty-four patients with RA and wasdesigned to determine the optimal total body dose of subcutaneouslyadministered D2E7. Patients were randomized to receive either 20, 40, or80 mg D2E7 or placebo weekly for twelve weeks, after which timeplacebo-treated patients were switched blindly to 40 mg D2E7/week.

Approximately 49% of patients reached ACR20 at 20 mg, 55% of patientsreached ACR20 at 40 mg, and 54% of patients reached ACR20 at 80 mg,while only 10% of patients receiving placebo reached ACR20 (set forth inFIG. 1A). Approximately 23% of patients reached ACR50 at 20 mg, 27% ofpatients reached ACR50 at 40 mg, and 20% of patients reached ACR50 at 80mg, and only 2% of patients receiving placebo reached ACR50. These dataillustrate that subcutaneous D2E7, particularly at a dose of 40 mg/week,generates a good response.

Example 3 Biweekly, Subcutaneous Administration of an Anti-TNFα AntibodyBiweekly, Subcutaneous Administration of D2E7

The clinical effects, safety, immunogenicity, and tolerance of RApatients with partial responses to MTX following every other weeksubcutaneous (s.c.) injections of placebo or D2E7 at several dose levelsfor up to twenty-four weeks in conjunction with continued MTX treatmentwas investigated.

Study Design

A placebo-controlled, double-blind, randomized, multi-center study inpatients with RA, who had insufficient efficacy or tolerability to MTXwas performed. During the course of the trial, patients were continuedon a stable dose of MTX with dose ranges specified in the inclusioncriteria described below.

This study consisted of two portions: 1) a “wash-out period” of fourweeks prior to the administration of the first dose medication, duringwhich time DMARDs (except for MTX) were withdrawn; and 2) a “placebocontrolled period” during which time patients were randomized to one offour cohorts of sixty-seven patients to receive placebo, 20, 40, or 80mg D2E7 (as a total body dose) given every other week s.c. for up to 24weeks. Each dose of study drug was administered as two s.c. injectionsof 1.6 mL each. The patient's first dose was administered by medicalpersonnel as part of the patient's training. Subsequent doses wereself-administered by the patient at the study under the directobservation of trained personnel for the first four weeks. Thereafter,doses were administered outside the study site by the patient, a trainedindividual designated by the patient, or by medical personnel.Medication for four or five weeks was dispensed after each clinicalassessment. Patients were serially examined in weeks one, two, three,four, six, eight, twelve, sixteen, twenty, and twenty-four of the studywith the joint examinations being performed by a blinded assessor,independent of the treating physician.

This study enrolled two hundred seventy-one patients with RA. The studypopulation was representative of the moderate to severe RA population inNorth America: approximately 70% female, and predominantly over the ageof forty. The population was selected using predetermined inclusion andexclusion criteria, known to those of skill in the art e.g., a patientmust have received a diagnosis of RA as defined by the 1987-revisedAmerican College of Rheumatology (ACR) criteria (set forth in AppendixA)

Results

FIGS. 1B and 2-4 indicate that subcutaneous, biweekly D2E7 treatmentcombined with methotrexate was significantly better than placebo inreducing the signs and symptoms of RA at twenty-four weeks. All threedoses of D2E7 were statistically significantly more effective thanplacebo given weekly. Furthermore, D2E7 at 40 mg and 80 mg had betterefficacy than the 20 mg dose.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

ACR Definition of RA

The 1987 classification tree criteria and functions for rheumatoidarthritis (RA)

CRITERION DEFINITION 1. Arthritis of 3 or more joint areas At least 3joint areas simultaneously have had soft tissues swelling or fluid (notbony overgrowth alone) observed by a physician. The 14 possible jointareas are right or left PIP, MDP, wrist, elbow, knee, ankle, and MTPjoints. 2. Arthritis of hand joints Soft tissue swelling or fluid (notbony overgrowth alone) of Wrist the specified area observed by aphysician. Where 2 MCP areas are specified, involvement must have beenMCP or wrist simultaneous. MCP and wrist 3 Symmetric swelling(arthritis) Simultaneous involvement of the same joint areas (as definedin 1 on both sides of the body (bilateral involvement of PIPs. MCP's, orMTPs is acceptable without absolute symmetry) 4. Serum rheumatoid factorDemonstration of abnormal amounts of serum rheumatoid factor by anymethod for which the result has been positive in <5% of normal controlsubjects 5. Radiographic changes of rheumatoid arthritis Radiographicchanges typical of rheumatoid arthritis on posteroanterior hand andwrist radiographs which must include erosions or unequivocal bonydecalcification localized in or most marked adjacent to the involvedjoints (Osteoarthritis changes alone do not qualify) A patient is saidto have RA if he/she is included in 1 of the 5RA subsets listed in Table7 and has a clinical diagnosis of RA by his/her physician. Criteria 1,2, and 3 must have been present for at least 6 weeks. Arthritis andRheumatism, Vol. 31, No. 3 (March 1988)

APPENDIX A SEQ ID NO: 1: Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu SerAla Ser Val Gly1               5                   10                  15 Asp Arg ValThr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr            20                  25                  30 Leu Ala Trp TyrGln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile        35                  40                  45 Tyr Ala Ala Ser ThrLeu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly    50                  55                  60 Ser Gly Ser Gly Thr AspPhe Thr Leu Thr Ile Ser Ser Leu Gln Pro65                  70                  75                  80 Glu AspVal Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr                85                  90                  95 Thr Phe GlyGln Gly Thr Lys Val Glu Ile Lys             100                 105 SEQID NO: 2: Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro GlyArg 1               5                   10                  15 Ser LeuArg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr            20                  25                  30 Ala Met His TrpVal Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val        35                  40                  45 Ser Ala Ile Thr TrpAsn Ser Gly His Ile Asp Tyr Ala Asp Ser Val    50                  55                  60 Glu Gly Arg Phe Thr IleSer Arg Asp Asn Ala Lys Asn Ser Leu Tyr65                  70                  75                  80 Leu GlnMet Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                85                  90                  95 Ala Lys ValSer Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly            100                 105                 110 Gln Gly Thr LeuVal Thr Val Ser Ser         115                 120 SEQ ID NO: 3: GlnArg Tyr Asn Arg Ala Pro Tyr Xaa 1               5

APPENDIX B SEQ ID NO: 4: Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Xaa 1              5                   10 Xaa of SEQ ID NO: 4 = Tyr or AsnSEQ ID NO: 5: Ala Ala Ser Thr Leu Gln Ser 1               5 SEQ ID NO:6: Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val Glu1                5                  10                  15 Gly SEQ IDNO: 7: Arg Ala Ser Gln Gly Ile Arg Asn Tyr Leu Ala1                5                  10 SEQ ID NO: 8: Asp Tyr Ala Met His1               5 SEQ ID NO: 9: Asp Ile Gln Met Thr Gln Ser Pro Ser SerLeu Ser Ala Ser Ile Gly1                5                  10                  15 Asp Arg ValThr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr            20                  25                  30 Leu Ala Trp TyrGln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile        35                  40                  45 Tyr Ala Ala Ser ThrLeu Gln Ser Gly Val Pro Ser Arg Ile Ser Gly    50                  55                  60 Ser Gly Ser Gly Thr AspIle Thr Leu Thr Ile Ser Ser Leu Gln Pro65                  70                   75                  80 Glu AspVal Ala Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser Ala Pro Tyr                85                   90                  95 Ala Ile GlyGln Gly Thr Lys Val Glu Ile Lys             100                  105 SEQID NO: 10: Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro GlyArg 1               5                   10                  15 Ser LeuArg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr            20                  25                  30 Ala Met His TrpVal Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val        35                  40                  45 Ser Ala Ile Thr TrpAsn Ser Gly His Ile Asp Tyr Ala Asp Ser Val    50                  55                  60 Glu Gly Arg Phe Ala ValSer Arg Asp Asn Ala Lys Asn Ala Leu Tyr65                  70                  75                  80 Leu GlnMet Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys                85                  90                  95 Thr Lys AlaSer Tyr Leu Ser Thr Ser Ser Ser Leu Asp Asn Trp Gly            100                 105                 110 Gln Gly Thr LeuVal Thr Val Ser Ser         115                 120 SEQ ID NO: 11: GlnLys Tyr Asn Ser Ala Pro Tyr Ala 1               5 SEQ ID NO: 12: Gln LysTyr Asn Arg Ala Pro Tyr Ala 1               5 SEQ ID NO: 13: Gln Lys TyrGln Arg Ala Pro Tyr Thr 1               5 SEQ ID NO: 14: Gln Lys Tyr SerSer Ala Pro Tyr Thr 1               5 SEQ ID NO: 15: Gln Lys Tyr Asn SerAla Pro Tyr Thr 1               5 SEQ ID NO: 16: Gln Lys Tyr Asn Arg AlaPro Tyr Thr 1               5 SEQ ID NO: 17: Gln Lys Tyr Asn Ser Ala ProTyr Tyr 1               5 SEQ ID NO: 18: Gln Lys Tyr Asn Ser Ala Pro TyrAsn 1               5 SEQ ID NO: 19: Gln Lys Tyr Thr Ser Ala Pro Tyr Thr1               5 SEQ ID NO: 20: Gln Lys Tyr Asn Arg Ala Pro Tyr Asn1               5 SEQ ID NO: 21: Gln Lys Tyr Asn Ser Ala Ala Tyr Ser1               5 SEQ ID NO: 22: Gln Gln Tyr Asn Ser Ala Pro Asp Thr1               5 SEQ ID NO: 23: Gln Lys Tyr Asn Ser Asp Pro Tyr Thr1               5 SEQ ID NO: 24: Gln Lys Tyr Ile Ser Ala Pro Tyr Thr1               5 SEQ ID NO: 25: Gln Lys Tyr Asn Arg Pro Pro Tyr Thr1               5 SEQ ID NO: 26: Gln Arg Tyr Asn Arg Ala Pro Tyr Ala1               5 SEQ ID NO: 27: Ala Ser Tyr Leu Ser Thr Ser Ser Ser LeuAsp Asn 1               5                   10 SEQ ID NO: 28: Ala SerTyr Leu Ser Thr Ser Ser Ser Leu Asp Lys1               5                   10 SEQ ID NO: 29: Ala Ser Tyr LeuSer Thr Ser Ser Ser Leu Asp Tyr 1               5                   10SEQ ID NO: 30: Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Asp1               5                   10 SEQ ID NO: 31: Ala Ser Tyr LeuSer Thr Ser Phe Ser Leu Asp Tyr 1               5                   10SEQ ID NO: 32: Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu His Tyr1               5                   10 SEQ ID NO: 33: Ala Ser Phe LeuSer Thr Ser Ser Ser Leu Glu Tyr 1               5                   10SEQ ID NO: 34: Ala Ser Tyr Leu Ser Thr Ala Ser Ser Leu Glu Tyr1               5                   10 SEQ ID NO: 35: Val Ser Tyr LeuSer Thr Ala Ser Ser Leu Asp Asn 1               5                   10SEQ ID NO: 36: GACATCCAGA TGACCCAGTC TCCATCCTCC CTGTCTGCAT CTGTAGGGGACAGAGTCACC 60 ATCACTTGTC GGGCAAGTCA GGGCATCAGA AATTACTTAG CCTGGTATCAGCAAAAACCA 120 GGGAAAGCCC CTAAGCTCCT GATCTATGCT GCATCCACTT TGCAATCAGGGGTCCCATCT 180 CGGTTCAGTG GCAGTGGATC TGGGACAGAT TTCACTCTCA CCATCAGCAGCCTACAGCCT 240 GAAGATGTTG CAACTTATTA CTGTCAAAGG TATAACCGTG CACCGTATACTTTTGGCCAG 300 GGGACCAAGG TGGAAATCAA A 321 SEQ ID NO: 37: GAGGTGCAGCTGGTGGAGTC TGGGGGAGGC TTGGTACAGC CCGGCAGGTC CCTGAGACTC 60 TCCTGTGCGGCCTCTGGATT CACCTTTGAT GATTATGCCA TGCACTGGGT CCGGCAAGCT 120 CCAGGGAAGGGCCTGGAATG GGTCTCAGCT ATCACTTGGA ATAGTGGTCA CATAGACTAT 180 GCGGACTCTGTGGAGGGCCG ATTCACCATC TCCAGAGACA ACGCCAAGAA CTCCCTGTAT 240 CTGCAAATGAACAGTCTGAG AGCTGAGGAT ACGGCCGTAT ATTACTGTGC GAAAGTCTCG 300 TACCTTAGCACCGCGTCCTC CCTTGACTAT TGGGGCCAAG GTACCCTGGT CACCGTCTCG 360 AGT 363

What is claimed:
 1. A syringe pre-filled with a composition comprising40 mg of a human anti-TNFα antibody and a pharmaceutically acceptablecarrier, excipient or diluent, wherein the anti-TNFα antibody comprisesan IgG1 heavy chain constant region; a variable light (“V_(L)”) chainregion comprising a CDR1 having the amino acid sequence of SEQ ID NO:7,a CDR2 having the amino acid sequence of SEQ ID NO:5, and a CDR3 havingthe amino acid sequence of SEQ ID NO:3; and a variable heavy (“V_(H)”)chain region comprising a CDR1 having the amino acid sequence of SEQ IDNO:8, a CDR2 having the amino acid sequence of SEQ ID NO:6 and a CDR3having the amino acid sequence of SEQ ID NO:4.
 2. The syringe of claim1, wherein the V_(L) chain region of the anti-TNFα antibody has theamino acid sequence of SEQ ID NO:1 and the V_(H) chain region of theanti-TNFα antibody has the amino acid sequence of SEQ ID NO:2.